Effect of Dietary Magnesium on Development of

Atherosclerosis in Cholesterol-fed Rabbits

Yasuyoshi Ouchi, Rikaru E. Tabata, Konstantinos Stergiopoulos,
Fumiyasu Sato, Akinori Hattori, and Hajime Orimo

The effect of dietary magnesium (Mg) on the development of atherosclerosis In

cholesterol-fed rabbits was investigated. Male New Zealand White rabbits (n=31)
were placed on five kinds of diets: regular, 1% cholesterol, and 1 % cholesterol diets
supplemented with either 300, 600, or 900 mg (as Mg) of Mg sulfate. The regular and
1% cholesterol diets contained 400 mg of Mg per 100 g. Each rabbit received 100 g
daily of the appropriate diet Additional Mg was well tolerated and did not affect blood
pressure or body weight The rabbits were sacrificed after 10 weeks, and the oil red
O-posrave atherosclerotic area that covered the aortic Intima and the cholesterol
content of the aorta was measured. Additional Mg decreased both the area of the
aortic lesions and the cholesterol content of the aortas In a dose-dependent manner.
The 1 % cholesterol diet significantly Increased plasma cholesterol and triglycerlde
concentrations and decreased high density llpoprotein (HDL) cholesterol concentra-
tion. Additional Mg had no further effect on cholesterol and HDL cholesterol
concentrations, but it slightly decreased the rise In triglycerlde concentration. These
results Indicate that dietary Mg prevents the development of atherosclerosis In
cholesterol-fed rabbits by Inhibiting llpld accumulation In the aortic wall.
(Arteriosclerosis 10:732-737, September/October 1990)

E vidence that the hardness of drinking water inversely

correlates with the rate of mortality from apoplexy in
various areas of Japan was first provided by Kobayashi.1
results indicating that dietary Mg exerts an anti-
atherogenic action are scanty.
The purpose of this study was to investigate the effect
Schroeder and Brattleboro2 also showed that the annual of supplementary dietary Mg on the development of
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death rates from atherosclerotic heart disease and cere-
brovascular disease were higher in areas of the United
States where drinking water was not as hard. Similar
findings were also reported in the United Kingdom.3
Hardness of water is determined by both calcium (Ca)
and magnesium (Mg) concentration. As the influence of
Ca on water hardness is greater than that of Mg, the
beneficial effect of hard drinking water had been attrib-
uted to the effect of Ca. Karppanen et al., 4 however,
suggested that Mg contributes to the decrease in mortal-
ity rate from ischemic heart disease (IHD). They showed
that in various countries the ratio of Ca to Mg in the diet
significantly correlated with the mortality from IHD. Fur-
thermore, cardiovascular mortality has been found to
correlate inversely with urinary excretion of Mg. These
epidemiologic studies clearly indicate that Mg intake may
inhibit the development of atherosclerosis. Actually, a
Mg-deficient, high cholesterol ( 1 % to 3%) diet reportedly
has caused more extensive lipid deposition in the aortas
of monkeys and rats. However, concrete experimental
From the Department of Geriatrics, Faculty of Medicine, Uni-
versity of Tokyo, Tokyo, Japan.
This study was supported by Grant-in-Aid for Scientific
Research No. 01570472 from the Ministry of Education, Science,
and Culture of Japan.
Address for correspondence: Yasuyoshi Ouchl, M.D., Depart-
ment of Geriatrics, Faculty of Medicine, University of Tokyo,
7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Received December 26, 1989; revision accepted March 23,
1990.
atherosclerosis in cholesterol-fed rabbits. Cholesterol
feeding in rabbits has been widely used as an experi-
mental model to investigate the effect of various agents
on the development of atherosclerosis.8-13
Methods
Animal Experiments
Thirty-one male New Zealand White rabbits weighing
about 2.5 kg each were purchased from the Saitama
Animal Laboratory (Saitama, Japan). They were kept
individually in stainless steel cages in a room where
temperature was maintained at 23°C. The rabbits were
divided into five groups and were put on five kinds of
diets: 1) a regular diet (n=6), 2) a 1% cholesterol diet
(n=6), 3) a 1 % cholesterol plus 0.3% Mg diet (n=6), 4) a
1% cholesterol plus 0.6% Mg diet (n=7), and 5) a 1%
cholesterol plus 0.9% Mg diet (n=6). Cholesterol, Mg
sulfate, or both were added to the regular diet (RC4,
Oriental Yeast, Tokyo, Japan), which contained 3% fat.
Since the regular diet and 1% cholesterol diet contained
0.4% Mg, diets 3, 4, and 5 actually contained a total of
700, 1000, or 1300 mg, respectively, of Mg per 100 g.
These diets contained 1360 mg of Ca per 100 g. The
other minerals in 100 g of diet were: 0.54 g phosphorus,
0.16 g sodium, 1.82 g potassium, 24.2 mg iron, 14.5 mg
aluminium, 1.01 mg copper, 4.49 mg zinc, 0.07 mg
cobalt, and 6.43 mg manganese. The calorie count was
294 kcal per 100 g. The rabbits were allowed free access
to tap water, which contained less than 1 mg/dl of Ca and

732
 MAGNESIUM PREVENTS ATHEROSCLEROSIS IN RABBITS
less than 2 mg/dl of Mg. Each rabbit received 100 g daily
of the assigned diet and was fed the whole diet every day
of the experiment. Diets containing additional Mg were
well tolerated; no abnormal physical signs such as weight
loss, decreased appetite, or diarrhea were noted during
the experiment.
Systolic blood pressure was measured once a week by
a device (Oiso Ikakikai, Tokyo, Japan) developed by
Kawaguchi14 and Grant and Rothschild.13 An artery in the
ear was compressed with an air-tight pressure capsule,
which was connected to a sphygmomanometer. The
level at which blood flow resumed during deflation of the
air-tight pressure capsule was considered to be systolic
blood pressure. We tested the accuracy of the noninva-
sive method for blood pressure measurement in three
male New Zealand White rabbits weighing approximately
3 kg. After the rabbits were anesthetized with pentobar-
bital, the right femoral artery was cut open, and PE-90
tubing was inserted into the abdominal aorta. The tubing
was connected to a strain-gauge transducer (Statham
P10EZ, Gould, Oxnard, CA) to measure arterial pressure.
The right femoral vein was also cannulated with PE-90
tubing. Blood pressure was increased by intravenous
infusion of norepinephrine (Sankyo, Tokyo, Japan) and
was decreased by intravenous infusion of nitroglycerine
(Nihon Kayaku, Tokyo, Japan) over the range of 60 to
145 mm Hg (by the noninvashve method). Arterial pres-
sure was measured simultaneously. Blood pressure mea-
sured by the noninvasive method was well correlated with
systolic arterial pressure (r=0.82, n=24, p<0.01); with
mean arterial pressure (r=0.88, n=24, p<0.01); and also
with diastolic pressure (r=0.93, n=24, p<0.01). Body
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weight was measured when blood pressure was taken.
At 10 weeks into the study, 10 ml of blood was
collected from the ear vein of each animal into a plastic
syringe containing 0.13 ml of ethylenediaminetetraacetic
add disodium solution (0.5 M). An additional 10 ml of
blood was collected into a plastic syringe, which did not
contain anticoagulants. These blood samples were cen-
trifuged at 1500 g for 15 minutes at 4°C. The plasma and
serum were stored at -20°C until chemical analysis was
performed. After blood sampling, rabbits were exsan-
guinated under pentobarbital anesthesia.
The total cholesterol concentrations and the high den-
sity lipoprotein (HDL) cholesterol concentrations in the
plasma were measured with an enzymatic method and a
heparin-Mn++ precipitation-enzymatic method, respec-
tively. The plasma triglyceride concentrations were mea-
sured with an enzymatic method. The total protein, Mg,
and inorganic phosphorus concentrations in serum were
measured with an auto-analyzer (Hitachi 736, Tokyo,
Japan). Ca concentration in serum was measured with
atomic absorption spectrophotometry (Hitachi 180-60).
Quantifying Atherosclerotic Plaque In
Aortic Intlma
Aortas were carefully removed from the aortic root to
the bifurcation. The surrounding adventitial tissues were
cleaned, and the entire aortas were washed twice with
saline. The aortas were longitudinally divided with sharp
scissors into anterior and posterior halves of approxi-
Ouchi et al. 733
mately the same size. The posterior half of each aorta was
affixed to a plastic board and was fixed by incubation in
3.5% formaldehyde solution (pH 7.0, Muto Pure Chemi-
cals, Tokyo, Japan) for 24 hours. The aortas were then
washed with distilled water and stained by incubation in
oil red O solution for 20 minutes, as described by Willis et
al. 10 Oil red O solution was prepared as follows: Oil red O
(Nacalai Tesque, Kyoto, Japan) was dissolved in isopro-
pyl alcohol to a concentration of 5 mg/ml. Six aliquots of
the solution were subsequently diluted by adding four
aliquots of distilled water. After staining, the intimal sur-
face was photographed. The area of the stained intimal
surface was measured with an x-y digitizer connected to
a microcomputer (PC-9801 VM2, Nihon Electrics, Tokyo,
Japan). The ratio of the stained area to the whole intimal
surface of the posterior half of the aorta was considered
to be the magnitude of atherosclerosis.
Measurement of Cholesterol, Calcium, and
Magnesium In Aortic Wall
Cholesterol content was measured in the anterior half
of the aorta. The aorta was freeze-dried and weighed.
Cholesterol was extracted from the freeze-dried aorta by
incubating it in 3 ml of chloroform/methanol (2:1) solution
at 4°C for 15 hours by the method described by Hollander
et al. 18 Cholesterol was further extracted by incubating
the aorta in 3 ml of the same solution at 50°C for
20 minutes. The two aliquots of solution were mixed, and
1 ml of the solution was evaporated with nitrogen gas.
The residual substance was dissolved in isopropyl alco-
hol/Triton X-100 (10%) solution. Cholesterol concentra-
tion was measured for the solution by the enzymatic
method developed by Allain et al. 17
The aorta was subsequently homogenized with a poly-
toron homogenizer, and the Ca and Mg concentrations in
the homogenate were measured with atomic absorption
spectrophotometry (Hitachi 180-60).
Statistics
The data were analyzed by using one-factor analysis of
variance. If a statistically significant effect was found, the
Newman-Keuls test was performed to isolate the differ-
ences between groups. Student's f test for paired data was
performed to test the significance of blood chemical data
before and after the experiment A p value of less than 0.05
was considered significant. All data are presented in the
text, tables, and figures as the means±SEM.
Results
Blood Pressure, Body Weight, and
Blood Chemistry
Systolic blood pressures and body weights during the
experiment are shown in Figure 1. A 1% cholesterol diet
with additional Mg did not affect the time course of blood
pressure and body weight in the rabbits.
The results of plasma lipid concentration are shown in
Table 1. The 1% cholesterol diet significantly increased
the total cholesterol concentration in the plasma (regular
diet group, 43.7±3.8 mg/dl; 1% cholesterol diet group,
1233.3±89.5 mg/dl, p<0.01). Additional dietary Mg had
 734 ARTERIOSCLEROSIS VOL 10, No 5, SEPTEMBER/OCTOBER 1990

3-

2.5- O—-O Control (n»6)

• • 1 X chd (n=6)
m ik—A 1 % chol + Mg300»»(n-6)
X K 1* chol + Mg600»B(n-7)
2- D D 1 * chol + Mg900»9(n-6)

8 10 week

100-

I 90-
m 80-
(0

70-.
1 8 10 week
Figure 1 . The time-related changes in body weight (upper panel) and systolic blood pressure (lower
panel) during the experiment. No significant differences were observed among the five groups of
rabbits throughout the experiment. n=number of rabbits, chol=cholesterol, Mg=magneslum,
BW=body weight, SBP=systolic blood pressure.

no further effect. The 1% cholesterol diet also increased 1 % cholesterol diet significantly increased the cholesterol
triglyceride (TG) concentration in the plasma, and addi- content of the aorta: regular diet group, 7.7±0.4 mg/g dry
tional dietary Mg decreased the plasma TG concentration weight (DW); 1% cholesterol diet group, 70.7±6.1 mg/g
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that was increased by the 1% cholesterol diet. On the
other hand, the 1% cholesterol diet significantly
decreased plasma HDL cholesterol concentration (regu-
lar diet group, 25.0±1.0 mg/dl; 1% cholesterol diet
group, 12.0±0.6 mg/dl, p<0.01). Again, additional
dietary Mg had no further effect.
As shown in Table 2, additional dietary Mg increased
the serum Mg concentration, indicating effective absorp-
tion of Mg. No significant differences were observed in
the total protein concentration in the serum from all
groups of rabbits either before or after the experiments.
Similar results were obtained for serum Ca concentration,
except when the highest dose of Mg was given.
Atherosclerotic Plaque In Intimal Surface
of Aorta
As shown in Figure 2, the 1% cholesterol diet signifi-
cantly increased the oil red 0-positive area in the intimal
surface of the aortas (regular diet group, 3.2 ±1.4%;
1% cholesterol diet group, 37.3±1.3%, p<0.01). Although
the addition of 0.3% Mg had no effect, the addition of 0.6%
Mg and 0.9% Mg significantly reduced the area to 11.7±
0.9% (p<0.01 vs. 1% cholesterol diet group) and 9.2±
1.5% (p<0.01 vs. 1% cholesterol diet group), respectively.
Cholesterol, Calcium, and Magnesium Contents
In Aortic Wall
The cholesterol in the aortic wall was similar to that of
the atherosclerotic plaque. There was a significant corre-
lation (r=0.86, n=31, p<0.01). As shown in Figure 3, the
DW, p<0.01. The addition of 0.3% Mg once again had no
effect The addition of 0.6% and 0.9% Mg, however,
significantly reduced the cholesterol content to 42.6±7.3
mg/g DW (p<0.05 vs. 1% cholesterol diet group) and
31.2±2.7 mg/g DW (p<0.05 vs. 1% cholesterol diet
group), respectively.
The results of aortic Mg and Ca content are shown in
Table 3. The aortic Mg content was similar for the five
groups of rabbits. However, aortic Ca content tended to
decrease in these groups, although no statistical signifi-
cance was observed.
Discussion
In the present study, we demonstrated that dietary Mg
suppresses the development of atherosclerotic plaque in
the intimal surface of the aortas of rabbits on high choles-
terol diets. This result strongly suggests that dietary Mg
has an antiatherogenic effect; this would support the
epidemiological observations that suggest that Mg in-
take helps decrease mortality from atherosclerotic
diseases.1 ** Antiatherogenic action was not the result of
the effect of Mg on blood pressure or body weight. These
parameters were similar in the five groups of rabbits
throughout the experiment irrespective of the diet on
which they were placed. In the present study, no hypoten-
sive action of Mg was noted, although Mg reportedly has
a hypertensive action in hypertensive subjects18 and in
spontaneously hypertensive rats. Dietary Mg might not
influence the blood pressure of normotensive rabbits.
 MAGNESIUM PREVENTS ATHEROSCLEROSIS IN RABBITS Ouchi et al. 735

Table 1. Plasma Upid Concentrations In Five Groups of Rabbits

Group n T-Chol HDL-Chol Triglyceride
Control
B 6 43.2±5.9 24.5±2.1 60.0±17.0
A 6 43.7±3.8 25.0±1.0 56.7±5.4
1%Chol
B 6 36.8±2.7 28.7±1.0 43.8±4.2
A 6 1233.3±89.5**tt 12.0±0.6**tt 186.0±18.2**tt
1%Chol+Mg300 mg
B 6 36.8±4.2 24.3±2.6 65.3±23.1
A 6 1143.3±217.0**tt 14.2±0.9*t 139.4±22.6**tt*
1% Choi+Mg 600 mg
B 7 48.2±5.6 27.6±3.3 56.4±5.4
A 7 1207.9±80.8** f r 17.0±2.4*t 99.8±6.4*tt«
1%Chol+Mg 900 mg
B 6 40.5±4.7 25.7±2.2 50.7±6.0
A 6 1038.0±75.2**tt 13.5±5.2** 127.5±4.6**tt*
Values are given as mg/dl and are the means±SEM.
B=before experiment, A=after experiment, T-Chol=total cholesterol, HDL-chol=high density llpoproteln
cholesterol.
*p<0.05, **p<001 vs. control group fed a regular diet; tp<0.05, ttp<0.01 vs. B; $p<0.05, #p<0.01 vs. 1%
cholesterol diet group.

Table 2. Chemical Analysis of Serum In Five Groups of Rabbits before and after Experiments
T-proteln Calcium Magnesium Phosphorus
Group n (9/dl) (mEq/l) (mg/dl) (mg/dl)
Control
B 6 5.6±0.2 6.7±0.2 2.73±0.08 5.73±0.28
A 6 6.6±0.5t 6.7±0.1 2.52±0.14 5.55±0.34
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1%Chol
B 6 6.1 ±0.1 6.7±0.1 2.25±0.18 6.45±0.36
A 6 7.4±0.3tt 6.9±0.2 2.27±0.04 5.77±0.43
1%Chol+Mg 300 mg
B 6 6.0±0.2 6.5±0.1 2.17±0.06 6.53±0.18
A 6 7.9±0.3tt 6.3±0.1 3.43±0.14*tt 6.25±0.52*
1%Chol+Mg 600 mg
B 7 6.1 ±0.1 6.5±0.2 2.85±0.08 7.23±0.25
A 7 7.0±0.1tt 6.5±0.1 5.44±0.18**tt 7.23±0.38**
1%Chol+Mg 900 mg
B 6 5.9±0.2 6.5±0.1 2.20±0.09 6.80±0.53
A 6 7.2±0.3tt 5.9±0.1**tt 6.00±0.44" t t 6.68±0.47**
Values indicate means±SEM.
B=before experiment, A=after experiment, T-protein=total protein, Chol=chcHesterol.
*p<0.05, **p<0.01 vs. control group fed a regular diet; tp<0.05, ttp<0.01 vs. B.

Some studies have been conducted on the effect of
dietary Mg on vascular lesions, rto et al. 20 reported that
the addition of Mg to diet effectively prevented the
development of coronary atherosclerosis induced by
vitamin D in pigs. Renaud et al. 21 reported that the
administration of Mg (356 mg per 100 g of diet) to rabbits
fed saturated fat slightly decreased both the atheroscle-
rotic lesions of the aortic intima and the cholesterol
content of the aorta. Although these results are basically
consistent with those we obtained in the present study,
dietary Mg seemed to have less of an effect than it did in
our present study. This might be because different doses
of Mg were administered and different methods were
used to evaluate the severity of atherosclerosis. In
Renaud's study, severity was semiquantitatively deter-
mined by visual grading. On the other hand, Nakamura et
al. 22 reported that a deficiency of dietary Mg resulted in
enhanced atheroma formation in the aortic intima of rats,
indicating the physiological significance of Mg intake.
The exact mechanism of the inhibitory effect of Mg on
the development of atherosclerosis is not yet known. In
the present study, dietary Mg significantly decreased
cholesterol content in the aorta without reducing total
cholesterol concentration in plasma. Moreover, Mg sup-
 736 ARTERIOSCLEROSIS V O L 10, No 5, SEPTEMBER/OCTOBER 1990

50 Table 3. Aortic Calcium and Magnesium Content in

Five Groups of Rabbits

40
u* t t p<0.01 vs. 1% Choi
M Group Aortic Ca Aortic Mg
mean±SE Control 6 551.5± 112.9 113.4±23.1
1%Chol 6 505.9 ± 173.9 138.3±20.3
30
1%Choi+Mg 300 mg 6 267.3±9.0 138.4±3.3
1%Choi+Mg 600 mg 7 332.7±14.3 138.8+6.5
20
1%Choi+Mg 900 mg 6 283.7+191.9 139.5±20.0
*tt Values are given as fig/mg dry weight and are the
10 means±SEM.
Chol=cholesterol, Mg=magnesium, Ca=calcium.
(6)
Control 1%Chol 1%Chol 1%Chol 1%Chol which protects against the invasion of LDL into the aortic
+ + + wall. As for the effect of Mg on vascular endothelial cells,
Mg 300mg 600mg 900mg increased production of prostacyclin, a known potent
Figure 2. The effect of magnesium (Mg) intake on the percent antithrombotic substance, has already been reported.27
area of atherosclerotic plaque that covered the aortic intimal
surface. *p<0.05, **p<0.01 vs. control group fed a regular diet,
Further studies will be required to determine the mecha-
ttp<0.01 vs. 1% cholesterol diet group. Chol=cholesterol. The nism. On the other hand, additional dietary Mg
numbers of experiments are shown in parentheses. decreased the plasma TG concentration that had been
increased by the 1 % cholesterol diet. Although the role of
150 TG in atherogenesis is still controversial, the possibility
that the decrease in plasma TG concentration may con-
t p<0.05 vs. \% Choi tribute to the suppression of the development of athero-
(n)
mean ± SE sclerosis in cholesterol-fed rabbits cannot be excluded.
100 The pharmacological basis of the inhibitory effect of
Mg on the development of atherosclerosis has not been
I** determined. However, Mg has been shown to modulate
Ca influx in vascular smooth muscles. Altura and Altura
50 l*,t found that acute reduction in extracellular Mg concentra-
, t tion increased the Ca content in vascular smooth mus-
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cles and that acute elevation had the opposite effect. In

(6)

" Control
(6)
1%Chol 1%Chol 1%Chol 1%Chol
m the present study, we also observed that dietary Mg
+ + + fed rabbits. Furthermore, the increase in extracellular Mg
Mg 300mg 600mg 900mg
Figure 3. The effect of magnesium (Mg) intake on the choles-
terol (chol) content of aortic wall. *p<0.05, **p<0.01 vs. control
group fed a regular diet; tp<0.05 vs. 1% cholesterol diet group.
The numbers of experiments are shown in parentheses.
plements did not further affect plasma HDL cholesterol
concentration, which was reduced when a 1 % cholesterol
diet was administered. These findings suggest that Mg
might prevent the development of atherosclerosis by
modulating cholesterol accumulation in the aortic wall.
Although the mechanism responsible for this has not yet
been clarified, a few possibilities exist. First, pathology
studies have revealed that many foam cells laden with
large amounts of lipids were clustered at the thickened
intima in the aorta of cholesterol-fed rabbits. 2122 Thus,
Mg possibly prevents the formation of foam cells in the
aorta of cholesterol-fed rabbits. Second, Mg may protect
the vascular endothelial cells; the presence of hypercho-
lesterolemia reportedly increases the permeability of the
aorta because of the endothelial cell damage in
cholesterol-fed rabbits. Since the vascular endothelial
cell layer acts to prevent the entry of macromolecules
such as low density lipoprotein (LDL) in the circulating
blood into blood vessels, Mg may preserve this barrier,
tended to decrease Ca content in the aorta of cholesterol-
concentration has been reported to decrease the influx of
s-
Ca into the aorta and portal veins of rats30 and also into
cultured rat aortic smooth muscle cells. The effect of
extracellular Mg is thought to result from the competition
with Ca at binding sites in vascular smooth muscle cell
membranes. In contrast, White and Hartzell used a
patch clamp technique to show that the increase in
intracellular free Mg exerted only a small effect on
voltage-dependent Ca current in isolated frog cardiac
myocytes. Thus, extracellular Mg could have Ca entry-
blocking action. This action is somewhat different from
that of Ca antagonists, because Mg acts on both voltage-
dependent and receptor-operated Ca channels, unlike
organic Ca antagonists, which have been thought to act
only on voltage-dependent Ca channels. The calcium
entry-blocking action of Mg may contribute to the sup-
pression of the development of atherosclerotic lesions; Ca
antagonists, such as nifedipine nicardipine, verap-
amil, and diltiazem, have reportedly suppressed the
development of atherosclerosis in cholesterol-fed rabbits.
Interestingly, lanthanum, a trivalent cation, has sup-
pressed the development of atherosclerosis in cholesterol-
fed rabbits.13 Lanthanum has been shown to block Ca
entry by occupying Ca binding sites34 in various types of
cell membranes, and the action is similar to that of Mg.
 MAGNESIUM PREVENTS ATHEROSCLEROSIS IN RABBITS
Solid evidence proving that dietary Mg can influence
the development of human atherosclerosis has not been
provided. The results of this study might not be applica-
ble to humans because the dose of Mg that proved
effective was much higher than the ordinary Mg intake in
humans. Moreover, the etiology of atherosclerosis might
differ in cholesterol-fed rabbits and in humans. Neverthe-
less, the role of Mg intake in the prevention of human
atherosclerosis should be further investigated, as the
beneficial effect of dietary Mg is important from a nutri-
tional standpoint.
In conclusion, we found that an increased amount of
dietary Mg suppressed the development of atheroscle-
rotic lesions in the aorta of cholesterol-fed rabbits without
affecting plasma total cholesterol and HDL cholesterol
concentrations. These findings support the results of
epidemiological studies.1-6 However, the mechanisms of
action and the clinical and nutritional implications should
be investigated further.
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